The detection of gene expression is paramount for the detection of the functioning of organisms in their respective environments. There is a great commercial interest in being able to detect the effects of certain environmental stimuli, such as environmental contaminations, on populations of cells in the environment (both aquatic and terrestrial environments). For example, there is great commercial interest in quantifying the effect of various types of commercial dumping in bodies of water, such as gulfs and oceans, on these ecosystems.
Specific applications of the measurement of gene expression include 1) the functioning of bacteria in fermentors for the production of biotechnological products; 2) the detection and measurement of microorganism activity in the environment, either for bioremediation, activity of genetically engineered organisms for agricultural, or pesticide activity; and 3) the measurement of activity of natural microbial components of ecosystems, including nitrogen fixing organisms, carbon dioxide fixing organisms (to assess the impact of greenhouse gases and their bioremediation), sulphate-reducing organisms, nitrate-reducers, nitrifiers etc.
An integral part of all gene expression methodologies is the isolation of mRNA. When a gene is active, the first process following gene activation is the synthesis of mRNA on a DNA template. This process is termed "transcription". If the mRNA is isolated and probed with a probe which is homologous to the mRNA, the total amount of mRNA can be quantified and the extent of expression of the target gene determined. Unlike the detection of activity for a particular enzyme, where there are different reagents, detection systems etc. which are needed, only one basic technology is needed to detect expression at the mRNA level of any gene in all organisms.
Currently, technology exists for the rapid isolation of mRNA from eucaryotic cells. This process involves guanidinium-isothiocyanate extraction followed by chromatography on polydT cellulose or some other support that takes advantage of the fact that all eucaryotic mRNA's have polyA tails.
No simple technology exists for the isolation of procaryotic (bacterial) mRNA because procaryotic mRNA lack polyA tails. Additionally, bacterial cell walls are more refractile than eucaryotic cells and require some degree of mechanical disruption. Thus, the present invention combines guanidinium-isothiocyanate extraction with mechanical disruption for RNA isolation from procaryotic cells.
Target gene mRNA isolation, detection, and quantification is not enough to determine the level of gene expression by specific microorganisms. For example, a high concentration of mRNA of a target gene may be only the result of a large concentration of cells. Prior art methods can only determine total mRNA content but cannot determine whether a single cell is producing a tremendous amount of mRNA or a whole population of cells is producing a very small amount mRNA. It is conceivable that a sample from an environment may yield a high target mRNA level because it has a very large quantity of cells with a low level of gene expression. Thus, it is essential to normalize the amount of mRNA present to the number of target cells. Typically, this has been done by normalizing mRNA to 23S rRNA content on gels. Although this may be an accurate method for pure cultures, this method will not function to provide useful data in mixed cultures or in the environment where a large amount of nontarget 23S rRNA will be present.
Various prior art methods have been used for the isolation of mRNA. For example, Pichard and Paul (1) disclosed the extraction of mRNA from procaryotic organisms using a modification of method of Chomczynski and Sacchi (2) combined with radio-labeled sense and antisense RNA probes to detect expression of specific genes in isolated indigenous populations of microorganisms. The reference does not disclose any further steps for combining the extraction of DNA from an additional aliquot of the same sample with sense or antisense RNA probe to quantitate expression of specific DNA sequences, target genes, and from this, information determining the amount of specific RNA expressed per target gene DNA in a mixed cell population.
Rymaszewski et al. (3) discloses a method of measurement of DNA content in cell lysates prepared for subsequent isolation of mRNA from the same sample of eucaryotic cell lines or cell cultures. The total DNA content is determined in the quantitation of cellular mRNA in relation to total cellular DNA content is made. The reference does not mention the determination of the level of the specific mRNA in relation to its specific DNA sequence (gene dose).
Raha et al. (4) discloses a method for the simultaneous extraction of total cellular RNA and DNA from the same sample as do the Karlinsey et al. reference (5) and the Zarkenga and Gamble reference (6). The isolated RNA and DNA is suitable for dot blot analysis. Neither reference shows or suggests the determination of the level of specific mRNA expression in relation to its specific DNA sequence by the use of radio-labeled sense or antisense RNA probes.
Atlas and Sayler (7) disclosed the direct extraction of DNA from environmental samples (i.e. mixed cell population) coupled with dot blot hybridization of radio-labeled DNA probe as a method for monitoring the presence of a specific DNA sequence, target gene, in the environment. The reference does not suggest or show monitoring of radio-labeled sense or antisense RNA probes to determine the level of mRNA expression of a specific DNA sequence.
The following references provide technical background for the present invention but are no more pertinent with regard to the patentability of the present invention than those references discussed above: Fleming, J.T. et al. (8); Tsai, Y-L et al. (9); Tsai, Y-L et al. (10); Oelmuller, U. et al. (11); and U.S. Pat. No. 5,021,335 to Tecott et al.
No prior art reference solves the problem of detecting gene expression in mixed cultures or in the environment which is adequate for the commercial applications discussed above. The present invention provides a novel solution to the problem by determining the target, or gene specific mRNA per target DNA or gene expression per gene dosage of sample. This is the most accurate measure of transcriptional activity in the gene expression providing significantly more useful information than the prior art methods set forth above.